In humans, four important glycoprotein hormone heterodimers (LH, FSH, TSH AND CG) have identical a subunits and differing .beta. subunits. Three of these hormones are present in virtually all other vertebrate species as well; CG has so far been found only in primates and in horse placenta and urine.
PCT application WO90/09800, published Sep. 7, 1990, describes a number of modified forms of these hormones. One important modification is C-terminal extension of the .beta. subunit by the carboxy terminal peptide of human chorionic gonadotropin or a variant thereof. Other muteins of these hormones are also described. In addition, U.S. Ser. No. 08/049,869 filed Apr. 20, 1993, the parent application herein, describes modifying these hormones by extension or insertion of the CTP at locations other than the C-terminus. The relevant positions for the CTP are from any one of positions 112-118 to position 145 of the .beta. subunit of human chorionic gonadotropin. The PCT application describes variants of the CTP extension obtained by conservative amino acid substitutions such that the capacity of the CTP to alter the clearance characteristics is not destroyed. The parent application additionally discloses fragments shorter than the sequence extending from positions 112-118 to 145.
The CTP-extended .beta. subunit of FSH is also described in two papers by applicants herein: LaPolt, P. S. et al.; Endocrinology (1992) 131:2514-2520 and Fares, F. A. et al.; Proc Natl Acad Sci USA (1992) 89:4304-4308. Both of these papers are incorporated herein by reference.
The crystal structure of the heterodimeric form of human chorionic gonadotropin has now been published in more or less contemporaneous articles; one by Lapthorn, A. J. et al. Nature (1994) 369:455-461 and the other by Wu, H. et al. Structure (1994) 2:545-558. The results of these articles are summarized by Patel, D. J. Nature (1994) 369:438-439.
At least one instance of preparing a successful single-chain form of a heterodimer is now known. The naturally occurring sweetener protein, monellin, is isolated from serendipity berries in a heterodimeric form. Studies on the crystal structure of the heterodimer were consistent with the proposition that the C-terminus of the B chain could be linked to the N-terminus of the A chain through a linker which preserved the spatial characteristics of the heterodimeric form. Such a linkage is advantageous because, for use as a sweetener protein, it would be advantageous to provide this molecule in a form stable at high temperatures. This was successfully achieved by preparing the single-chain form, thus impeding heat denaturation, as described in U.S. Pat. No. 5,264,558.
Published PCT application WO91/16922 published Nov. 14, 1991 describes a multiplicity of chimeric and otherwise modified forms of the heterodimeric glycoprotein hormones. In general, the disclosure is focused on chimeras of .alpha. subunits or .beta. subunits involving portions of various .alpha. or .beta. chains respectively. One construct simply listed in this application, and not otherwise described, fuses substantially all of the .beta. chain of human chorionic gonadotropin to the .alpha. subunit preprotein, i.e., including the secretory signal sequence for this subunit. This construct falls outside the scope of the present invention since the presence of the signal sequence intervening between the .beta. and .alpha. chains fails to serve as a linker moiety as defined and described herein.
It has now been found that the normally heterodimeric glycoprotein hormones retain their properties when in single-chain form, including single-chain forms that contain the various CTP extensions and insertions described above.